Allantoin is an important fine chemical that can be widely used in pharmaceutical, cosmetic and agricultural industries. Currently, allantoin is mainly produced by plant extraction or chemical synthesis. Due to the cost and environmental concerns, biosynthesis of allantoin from renewable feedstock is much more desirable. However, microbial production of allantoin from simple carbon sources has not yet been achieved so far. In this study, de novo biosynthesis of allantoin was achieved by constructing an artificial biosynthetic pathway. First, screening of efficient urate oxidases and xanthine dehydrogenases enabled allantoin production from hypoxanthine, a natural intermediate in purine metabolic pathway in Escherichia coli. Then, assemble of the entire pathway resulted in 13.9 mg/L allantoin from glucose in shake flask experiments. The titer was further improved to 639.8 mg/L by enhancing the supply of the precursor, redistribution of carbon flux, and reduction of acetate. Finally, scale-up production of allantoin was conducted in a 1-L fermentor under fed-batch culture conditions, which enabled the synthesis of 2360 mg/L allantoin, representing a 170-fold increase compared with the initial strain. This study not only demonstrates the potential for industrial production of allantoin, but also provides a bacterial platform for synthesis of other purines-derived high-value chemicals.